1a- and/or 7-substituted derivatives of mitomycin



June 17, 1969 MASANAQ T U ET AL 3,450,705

1a.- AND/OR 7-SUBSTITUTED DERIVATIVES OF MITOMYCIN Filed July 9, 1965 Sheet of e INVENTORS mnsmm 1 1/1750 ATTORNEYS June 17, 1969 MASANAO MATSUI ET AL 3,450,705 I 18- AND/0R 7-SUBSTITUTED DERIVATIVES OF MITOMYCIN Filed July 9, 1965 Sheet 2 of 6 ATTORNEYS June 17, 1969 le- AND/OR 7-SUBSTITUTED DERIVATIVES OF MITOMYCIN Filed July 9, 1965 MASANAO MATSUI ET AL Sheet 3 016 INVENTORS 3 n w H m; c 4T5 L i BY 30/ G Tram Wyk/ ATTORNEYS I June 17, 1969 ASANAO MATSUI ET AL Filed July 9, 1965 it AND/OR 7SUBSTITUTED DERIVATIVES OF MITOMYCIN sheet 5 are Ju ne 17, 1969 MASANAO MATSUl ET AL 3,450,705

11 AND/OR 7-SUBSTITUTED DERIVATIVES OF MITOMYCIN Filed July 9. 1965 sheet 6 of s ATTORNEY S United States Patent *ce Patented June 17, 1969 3,450,705 1a- AND/0R 7-SUBSTITUTED DERIVATIVES H N 0F MITOMYCIN Masanao Matsui, Yasuhiro Yamada, Tadashi Hirata, and

Shigetoshi Wakaki, Tokyo, Japan, assignors to Kyowa 5 H89 giakko Kogyo Co., Ltd., Tokyo, Japan, a corporation of apan Filed July 9, 1965, Ser. No. 470,849 Claims priority, application Japan, July 9, 1964, ito yci 0 Int Cl A61k 21/00 10 Genetically, these compounds correspond to the formula us. (:1. 260-295 8 Claims 0 011,0 0 ONE:

a ABSTRACT OF THE DISCLOSURE Hag Compounds of the formula N-"/ i X /CHZOCONH wherein:

pom A B 0 H 0 \N 00H OCH; NH2

5 ,1, a 8a a N C O R These compounds thus have the following skeleton: wherein X is amino, lower alkylamino, phenylamino or pyridyl and COR is halo (lower) alkanoyl, halobenzoyl, nitrobenzoyl, alkenoyl, acetylglycyl, sorbyl or acetylmethionyl and, when X is other than amino, COR is lower alkanoyl or benzoyl.

The present invention relates to new antibiotics, more particularly to new derivatives of mitosane compounds. and f called mltosane compounds' In 1956, Hata et a1. isolated new antibiotics from the Mltomycm and C are excelkfnt antlblotlgs but fermented broth of Streptomyces caespitosus and named they have a defecft m that F toxlc to human blood the antibiotics mitomycin A and B. In 1958, Wakaki et al. In order to obtam less toxlc mltosane compounds m isolated a third antibiotic from the same broth and named 40 research has been mducttd even bfore i determinatlon it mitomycin C. Since then it has been determined that of the chemlcal composmon of mliomycm B and C the chemical composition of the three compounds is as for example; methyl -Imtomyc1n or porfiromycm follows: was synthesized by reactmg mitomycln C and methyl iodide in the presence of potassium carbonate. After the determination of the chemical composition of mitomycin if A, B and C, research to obtain derivatives has been ac- CHOCONH tively conducted. For example, Belgian patent 624,559 H300 provides some 6,7 and la-substituted derivatives of mitw H C 00H; sane compounds.

3 The present invention is concerned with novel mitosane N o 0 I II NH derlvatlves of reduced toxlcity and of correspondingly enhanced utility as antibiotics, and to the preparation thereof. Mitomycm A The new antibiotics of the present invention are ex- 0 pressed by the following general formula:

CHQOOONH: 11,00 ornoooNH,

OH X H O pom 6O Ha Mitomycin B wherein X is OCH or amino, aliphatically monoor disubstituted amino (e.g. lower alkylamino such as methylamino, ethylamino, propylamino, isopropylamino, n-butylamino, etc. or di(lower) alkylamino such as diethylamino, dipropylamino, diisopropylamino, di-n-butylamino, etc.), aromatically monoor di-substituted amino (e.g. phenylarnino, diphenylamino, chlorophenylamino, bromophenylamino, methylphenylamino, dimethylphenylamino, phenylethylamino, etc.) or heterocyclic amino (e.g. pyridyl, etc.), COR is substituted acyl (e.g. halo (lower) alkylcarbonyl, dihalo-lower alkylcarbonyl, lower alkanoylamino(lower) alkylcarbonyl, lower alkanoyl(lower) alkylthio(lower) alkyleneamino(1ower)alkylcarbonyl, lower alkenyl(lower)alkenylcarbonyl, nitrophenylcarbonyl, dinitrophenylcarbonyl, halophenylcarbonyl, dihalo-phenylcarbonyl such as, inter alia, acetylglycyl, bromoacetyl, chloropropionyl, nitrobenzoyl, dinitrobenzoyl, sorbyl, acetylmethionyl, chlorobenzoyl, dichlorobenzoyl, iodobenzoyl, fluoroacetyl, dichloroacetyl, chloroacetyl) and COR can be unsubstituted acyl when X is substituted amino.

Compounds I are synthesized from coresponding mitosane compounds of the formula:

CH2OCONH2 wherein X has the same significances as in Formula I. When X is OCH the starting compound is mitomycin A and when X is NH the compound is a derivative of mitomycin C.

According to the present invention, the compounds I are produced by acylation of the starting mitosane compound, the acylation being carried out by reaction with an organic acid in the presence of a dehydrating condensing agent of the formula:

wherein R stands for an aliphatic or aromatic hydrocarbon radical, examples being dicyclohexylcarbodiimide and ditolylcarbodiimide, and the reaction being advantageously effected in a solvent for the starting mitosane compound.

The acylation is achieved by dissolving equimolar amounts of starting compound, organic acid and carbodiimide in a solvent therefor and allowing the resultant solution to stand. As the ensuing reaction progresses, the carbodiimide is converted to N,N'-dihexylurea and is precipitated:

Example 1.la-N-acetylglycylmitomycin C 2 g. (grams) of mitomycin C is dissolved in 120 m1. (milliliters) of dioxane, after which 80 mg. (milligrams) of N-acetylglycine in 20 ml. of water is added. 5 g. of dicyclohexylcarbodiimide is then added to the mixture,

which is then allowed to stand for 24 hours. The reaction mixture is filtered and the filtrate is concentrated in vacuo at low temperature. The concentrate is extracted with ether in order to exclude unreacted dicyclohexylcarbodiimide. The ether-insoluble part is dissolved in a mixture of acetone and ethyl acetate (1:1) and then chromatographed using silica-gel. From the second fraction, 1a-N-acetylglycylmitomycin C is obtained. Yield 800 mg.

Analysis.Calcd. for C H O N C, 52.65; H, 5.35; N, 16.16. Found: C, 52.65; H, 5.35.

The infra-red spectrum in Nujol is shown in FIG. 1.

Example 2.1a-bromacetylmitomycin C 1 g. of mitomycin C is dissolved in 60 ml. of anhydrous tetrahydrofuran. 455 mg. of bromacetic acid and 630 mg. of dicyclohexylcarbodiimide are added to the solution which is then allowed to stand for 1 hour. The reaction mixture is filtered and the filtrate is concentrated under reduced pressure at low temperature. Silica-gel chromatography is carried out using a mixture of acetone and ethyl acetate (3:7) as solvent. The purple first fraction is concentrated under reduced pressure, and the concentrate is dissolved in acetone and then ether added to effect precipitation of the objective la-bromacetylmitomycin C. Yield 950 mg.

Analysis.Calcd. for C H N O Br: C, 44.88; H, 4.18; N, 12.54. Found: C, 44.70; H, 4.10.

The infra-red spectrum in Nujol is shown in FIG. 2.

Example 3.-1a-a-chloropropionylmitomycin C 1 g. of mitomycin C is dissolved in 60 ml. of anhydrous dioxane. 330 mg. of a-chloropropionic acid in 20 ml. of dioxane and 630 mg. of dicyclohexylcarbodiimide are then added with stirring. The reaction is completed in 20 minutes. The reaction mixture is filtered and concentrated under reduced pressure at low temperature. Silica-gel chromatography is conducted using ethyl acetate-benzene (1:1) as solvent. The main purple fraction is concentrated in vacuo, yielding the objective la-a-chloropropionylmitomycin C. Yield 1.1 g.

Analysis.Calcd. for C H O N Cl: C, 50.10; H, 5.10; N, 13.21. Found: C, 50.10; H, 5.01.

The infra-red spectrum in Nujol is shown in FIG. 3.

Example 4.-1a-para-nitrobenzoylmitomycin C 1 g. of mitomycin C is dissolved in ml. of acetone and, after addition of 520 mg. of p-nitrobenzoic acid and 650 mg. of dicyclohexylcarbodiimide, allowed to stand for 30 minutes at room temperature with stirring. The reaction mixture is filtered and concentrated under reduced pressure. Silica-gel column chromatography is conducted using ethyl acetate as solvent. The first fraction is concentrated and 1a-p-nitrobenzoylmitomycin C is obtained. Yield 1.3 g.

Analysis.-Calcd. for C H O N C, 54.66; H, 4.38; N, 14.49. Found: C, 54.31; H, 4.69; N, 13.87.

The infra-red spectrum in Nujol is shown in FIG. 4.

Example 5.1a-3.S-dinitrobenzoylnfitomycin C 1 g. mitomycin C is dissolved in ml. of acetone, after which 560 mg. of 3,5-dinitrobenzoic acid in 5 ml. of acetone and 700 mg. of dicyclohexylcardbodiimide in 5 ml. of acetone are stirred in. The reaction continues for 5 minutes. The resultant dicyclohexylurea is removed by filtering the reaction mixture. The filtrate is concentrated under reduced pressure at low temperature. The concentration is conducted until the volume becomes 6 of the initial volume. The resulting crystals of 1a-3,5-dinitrobenzoylmitomycin C are separated by filtration and washed 3 to 4 times with ether. Yield 1.4 g.

Analysis.-Calcd. for C H O N C, 50.00; H, 3.82; N, 15.91. Found: C, 50.11; H, 3.61; N, 15.41.

The infra-red spectrum in Nujol is shown in FIG. 5.

1 g. of mitomycin C is dissolved in 100 ml. of tetrahydrofuran and, after addition of 700 mg. of dicyclohexylcarbodiimide and 600 mg. of sorbic acid, allowed to stand for 3 days at room temperature with stirring. The reaction mixture is filtered. The filtrate is concentrated under reduced pressure at low temperature. Silicagel column chromatography is conducted using ethyl acetate as solvent in order to remove unreacted material. Ethyl acetate solution of the resulting material is washed 3 times with 5% NaHCO and dried with Na SO and then is concentrated. The chromatography is repeated. The purple fraction is concentrated to give la-sorbylmitomycin C. Yield 400 mg.

Analysis.-Calcd. for C 'H N O C, 57.18; H, 5.50; N. 13.61. Found: C, 56.81; H, 5.63; N, 12.93.

The infra-red spectrum in Nujol is shown in FIG. 6.

Example 7.1a-acetylmethionylmitomycin C 1 g. of mitomycin C is dissolved in 80 ml. of tetrahydrofuran, after which there are added 700 mg. of dicyclohexylcarbodiimide in a small amount of tetrahydrofuran and 1.3 g of N-acetylmethionine in a small amount of methanol with stirring. The reaction mixture desired product, 1a-benzoyl-7a-ethylmitomycin C. Yield mg.

Analysis.-Calcd. for C H O N C, 62.09; H, 5.69; N, 11.65. Found: C, 61.41; H, 5.81; N, 11.03.

The infra-red spectrum in Nujol is shown in FIG. 8.

Example 9.-1a-p-nitrobenzoylmitomycin A 100 mg. of mitomycin A is dissolved in 10 ml. of acetone, followed by addition of 70 mg. of dicyclohexylcarbodiimide and 60 mg. of p-nitrobenzoic acid. The reaction is continued for 10 minutes. The reaction mixture is filtered and concentrated under reduced pressure at low temperature. Ether is added to the concentrate to effect precipitation. The ether is removed by decantation, and the precipitate is dissolved in small amount of ethyl acetate. Silica-gel chromatography is conducted using a mixture of ethyl acetate and benzene (1:1). The reddish first fraction is dried in vacuo at low temperature, to yield 1a-p-nitrobenzoylmitomycin A. Yield 110 mg.

Analysis.Calcd. for C H O N C, 56.65; H, 4.43; N, 10.88. Found: C, 56.43; H, 4.41; N, 10.33.

The infra-red spectrum in Nujol is shown in FIG. 17.

Example 10 The following derivatives are produced:

Name of derivative Molecular formula Reacting materials 1a-3,4-dichloro benzoylmitomycin C--. CnHioNsOcCh la-o-iodobenzoyl-mitomycin C CnH 1a-p-chlorobenzoyl-mitomycin O C22H2IN4OGC1 Mitomycin C, 3,4-diehlorobenzoic acid N O I. Mitomycin C, o-iodo-benzoic acid Mitomycin C p-chlorobeuzoic acid.

1afiuoroacety1-mitomycin C C H nN4OsF Mitomycin C, fluoroacetic acid 3 1a-trichloroacetyl-mitomycin O- CnH NiOsCl Mitomycin C, trichloroacet c ac1d 20 minutes... 1. Fig. 1a-dichloroacetyl-mitomycin C O11HIEN406C112 Mitomycin C, dlchlqroacetic acid 15 minutes 1.20 g. Fig. 16. 1a-a-cl loi)propionyl7N-phenyl-mito- CMHZfiNAOsCl 7N-phenyl-mitomycm O, a chloropropioru'c acid 30 minutes 800 mg Fig. 12.

mycin 1a-acetyl-7N-pridy1-mitomycin O C2zH2sN4Oa 7N -pyridyl-mitomycm O, acetic acid 2O mmutes- 850 mg Fig. 13.

is allowed to stand for a night and the resulting dicyclohexylurea is removed. The filtrate is concentrated under 40 as reduced pressure at low temperature. Silica-gel chromatography is conducted using ethyl acetate as solvent. The purple first fraction is concentrated under reduced pressure to give the objective product, la-acetylmethionylmitomycin C. Yield 250 mg.

Analysis.-Calcd. for C H N O C, 52.40; H, 5.50; N, 13.88. Found: C, 52.12; H, 5.81; N, 13.24.

The infra-red spectrum in Nujol is shown in FIG. 7.

Example 8.-1a-benzoyl-7a-ethylmitomycin C 30 mg. of 7a-ethylmitomycin C is dissolved in 10 ml. of acetone and, after the addition of 10 mg. of benzoic acid and 25 mg. of dicyclohexylcarbodiimide, allowed to stand for a night. The reaction mixture is filtered and concentrated under reduced pressure at low temperature. Silica-gel chromatography is conducted using ethyl acetate as solvent. The first fraction is concentrated to give the The new compounds I are less toxic than mitomycin C is shown in the following:

Table 2 shows exemplary minimum inhibition concentrations of derivatives of the present invention against various test microorganisms.

TABLE 2 (Minimum inhibition concentration) la-acetyl- 1a-bromla-B-chlorola-a-chlorola-benzoylglyeylacet propionylpropionyl- 7a-ethy1- mitomitomitomitomitomycin O mycin C mycin C mycin O mycin C Staphylococcus aureus 209 P 0. 39 0. 39 0. 0. 195 3.12 Sarcina lutea POI 1001 0. 195 0.39 0. 195 0. 195 6. 25 Bacillus subtzlis ATCC 6633-.- 0. 195 0. 39 0. 097 0. 195 3. 12 Salmonella typhi 379 1. 56 3. 12 1. 56 1. 56 25 Shigella flezuert Za 3196.. 0. 195 3. 12 0. 78 1. 56 0. 195 ebstella pueumo'hiae 0/10.- 0. 78 3. 12 1. 56 1. 56 50 Proteus X19 3. 12 6. 25 I. 56 3. 12 50 Escherichia coli K12 1. 56 6. 25 3. 12 3. 12 25 Pseudomonas aeruginooa 3 3. 12 6. 25 l. 56 3. 12 50 Vibrio comma 62 0. 097 0. 024 0. 012 0. 024 50 Mycobactcrium tuberculosis 607- 0. 39 0. 048 0. 048 0. 048 0. 78 Streptococcus haemolg ticus 68---" 0. 195 0. 097 0. 048 0. 048 0. 195 Streptococcus faecalis 5 0.39 0.39 0. 78 0. 39 6. 25 c 119 0. 024 0. 024 0. 012 0. 012 3. 12 O'oryhebactcrlum diphtheriae 92 0. 097 0. 048 0. 048 O. 048 0. 195

7 8 What is claimed is: 3,332,944 7/1967 Cosulich et a1. 260-2472 1a-N-acetylglycylmitomycin C. 2,583,425 1/ 1952 Hawley 16730 la-p-nitrobenzoylmitomycin A. OTHER REFERENCES 3. 1a-3,5-dinitrobenzoy1mitomycin C.

4, l rb l it i C Berger, Medlclnal Chemlstry, lntersclence, second ed. 5

. 1a-acetylmethionylmitomycin C. 5 960) p. 43.

. 1a-p-nitrobenzoylmitomycin C. 1a-a-chloropropionyl-7-N-phenylmitomycin C. HENRY ULES, P Examme 1a-acety1-7-N-pyridylmit0mycin C. ALAN L. ROTMAN, Assistant Examiner.

References Cited 10 US. Cl. X.R. UNITED STATES PATENTS 260-3263, 999

3,226,393 12/1965 Meyer et a1. 260-295 

